Many physiological functions are mediated through neuropeptide secretion from the central nervous system. Sexual differentiation, function and pubertal development are modulated through the effects of gonadotropin releasing hormone (GnRH). In children with cystic fibrosis (CF), alterations in puberty and fertility are well-described complications. Neurosecretion may be modulated by the cystic fibrosis transmembrane conductance regulator (CFTR), resulting in abnormal regulation of physiologic processes. The central hypothesis of this proposal is that defective expression and function of the CFTR in brain alters neuropeptide trafficking and secretion and leads to the pathobiology manifested in patients with CF. This grant proposal addresses this central hypothesis with a combination of electrophysiological, cellular and molecular biological techniques, using GnRH, syntaxin 1A and CFTR as model proteins whose expression has been confirmed within hypothalamic neurons. Control mechanisms of neurosecretion will be examined in the fetal brain, and GT1-7, a GnRH-expressing hypothalamic neuronal cell line.
The specific aims of this proposal are: 1) To characterize the CFTR, syntaxin 1A and GnRH physiologic relationship in cultured fetal hypothalamic neurons in order to provide mechanistic information regarding their co-expression. Fetal hypothalamic neurons from rat will be cultured in vitro to assess ontogeny of CFTR and syntaxin 1A expression as it may modulate GnRH secretion; 2) To determine whether CFTR within the neuron functionally regulates membrane trafficking. The interaction between CFTR and syntaxin-lA in endo- and exocytosis will be evaluated using the fetal hypothalamic and GT1-7 cells. Electrophysiological measures, including capacitance and fluorophore quantitative techniques will be used; and 3) To identify whether ablation of CFTR regulates neuronal secretion. The CFTR gene, mRNA and protein have been identified by reverse transcriptase polymerase chain reaction (rt-PCR), in situ hybridization (NISH) and Western blot from GT1-7 cells. Antisense oligodeoxyribonucleotides developed against the CFTR gene and inhibitors of CFTR C1 conductance will be screened for effects upon secretion of GnRH. Electrophysiological techniques will confirm the efficacy of such interventions. CFTR-dependent regulation of neurosecretory processes may lead to an understanding of common manifestations in children with CF and illustrate a novel mechanism of homeostatic control.
